Chung Hee Jung, Ko Du Young, Moon Hyo Jung, Jeong Byeongmoon
Department of Chemistry and Nanoscience, Ewha Womans University , 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Korea.
Biomacromolecules. 2016 Mar 14;17(3):1075-82. doi: 10.1021/acs.biomac.5b01694. Epub 2016 Mar 2.
There are four EF-hand polypeptides in calmodulin, a natural ubiquitous calcium binding protein that activates the enzymes involved in Ca(2+)-mediated signal transduction. An EF-hand polypeptide has six carboxylate functional groups in the middle loop region between two rigid polypeptides. In this study, a calcium binding polymer (CBP) with a structure of poly(L-alanine)-poly(L-alanine-co-L-glutamic acid)-poly(ethylene glycol)-poly(L-alanine-co-L-glutamic acid)-poly(L-alanine) (PA-PAE-PEG-PAE-PA; A11.1-A3.4E3.2-EG40.1-A3.4E3.2-A11.1) was synthesized by mimicking the EF-hand polypeptide. The 6-7 carboxylate functional groups from PAE are expected to form a binding site for Ca(2+). As the Ca(2+) bound to CBP, small changes in the circular dichroism spectra and (13)C NMR spectra were observed, indicating that Ca(2+) binding to CBP induced changes in the conformation of CBP. The binding constant of CBP to Ca(2+) was investigated by using the competitive binding of 2,2',2″,2‴-{ethane-1,2-diylbis[oxy(4-bromo-2,1-phenylene)nitrilo]} tetraacetic acid (5,5-Br2-BAPTA). The binding constant obtained with a CaLigator program by least-squares fitting of the absorbance profile as a function of Ca(2+) concentration was 5.1 × 10(5) M(-1), which was similar to that of calmodulin. The selectivity of CBP for metal ion binding was compared among Ca(2+), Cu(2+), and Zn(2+). The binding constant was obtained through a similar competitive binding study with murexide. The binding constants for Ca(2+), Cu(2+), and Zn(2+) were 7.0 × 10(5), 4.2 × 10(5), and 1.7 × 10(5) M(-1), respectively, indicating 2-4-fold higher selectivity of CBP for Ca(2+) compared to Cu(2+) and Zn(2+). The CBP has selectivity for Ca(2+), and binding affinity for Ca(2+) was similar to the biological Ca(2+) binding motif of calmodulin.
钙调蛋白中有四种EF-手型多肽，钙调蛋白是一种天然存在的普遍存在的钙结合蛋白，可激活参与Ca(2+)介导信号转导的酶。一个EF-手型多肽在两条刚性多肽之间的中间环区域有六个羧酸酯官能团。在本研究中，通过模拟EF-手型多肽合成了一种具有聚（L-丙氨酸）-聚（L-丙氨酸-co-L-谷氨酸）-聚（乙二醇）-聚（L-丙氨酸-co-L-谷氨酸）-聚（L-丙氨酸）（PA-PAE-PEG-PAE-PA；A11.1-A3.4E3.2-EG40.1-A3.4E3.2-A11.1）结构的钙结合聚合物（CBP）。预计来自PAE的6-7个羧酸酯官能团会形成一个Ca(2+)的结合位点。当Ca(2+)与CBP结合时，观察到圆二色光谱和(13)C NMR光谱有微小变化，表明Ca(2+)与CBP的结合诱导了CBP构象的变化。通过使用2,2',2″,2‴-{乙烷-1,2-二基双[氧基(4-溴-2,1-亚苯基)腈]}四乙酸（5,5-Br2-BAPTA）的竞争性结合来研究CBP与Ca(2+)的结合常数。通过用CaLigator程序对吸光度曲线作为Ca(2+)浓度的函数进行最小二乘拟合得到的结合常数为5.1×10(5) M(-1)，这与钙调蛋白的结合常数相似。比较了CBP对Ca(2+)、Cu(2+)和Zn(2+)的金属离子结合选择性。通过与紫脲酸铵进行类似的竞争性结合研究获得结合常数。Ca(2+)、Cu(2+)和Zn(2+)的结合常数分别为7.0×10(5)、4.2×10(5)和1.7×10(5) M(-1)，表明CBP对Ca(2+)的选择性比Cu(2+)和Zn(2+)高2-4倍。CBP对Ca(2+)具有选择性，并且对Ca(2+)的结合亲和力与钙调蛋白的生物Ca(2+)结合基序相似。
